Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair

Abstract The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164)....

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Published in:PNAS nexus Vol. 3; no. 7; p. pgae242
Main Authors: Shah, Ronak, Aslam, Muhammad Assad, Spanjaard, Aldo, de Groot, Daniel, Zürcher, Lisa M, Altelaar, Maarten, Hoekman, Liesbeth, Pritchard, Colin E J, Pilzecker, Bas, van den Berk, Paul C M, Jacobs, Heinz
Format: Journal Article
Language:English
Published: US Oxford University Press 01.07.2024
Subjects:
Anemia
Antigens
Biological, Health, and Medical Sciences
Care and treatment
Cell cycle
Crosslinking
Decision making
DNA repair
Embryo fibroblasts
Fanconi syndrome
Fanconi's anemia
Genetic aspects
Genotoxicity
Immunoprecipitation
Lysine
MSH2 protein
MSH6 protein
Mutants
Mutation
Physiological aspects
Proliferating cell nuclear antigen
Replication
Spectrometry
Ubiquitination
Netherlands
PCNA monoubiquitination
mismatch repair
replication stress
Fanconi anemia
interstrand crosslink
ISSN:2752-6542, 2752-6542
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Summary:Abstract The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1−/− mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg−/+ mice. A combined mutation (PcnaK164R/K164R; Fancg−/−) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.
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R.S., M.A.A., and A.S. contributed equally to this work.
Competing Interest: The authors declare no competing interests.
ISSN:2752-6542
2752-6542
DOI:10.1093/pnasnexus/pgae242